Neuroprotection by hepatic cells and hepatocyte secretory factors

ABSTRACT

The present invention relates to compositions and methods for neuroprotection. In particular, provided herein are compositions (e.g., hepatocyte secretory factors) for alleviating and/or protecting against neuronal damage (e.g., resulting from stroke), and methods of use thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present invention claims the benefit of U.S. Provisional PatentApplication Ser. No. 61/482,099, filed May 3, 2011, which isincorporated by reference in its entirety.

STATEMENT REGARDING FEDERAL FUNDING

This invention was made with government support under Grant NumberCBET0932131 awarded by the National Science Foundation. The governmenthas certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to compositions and methods forneuroprotection. In particular, provided herein are compositions (e.g.,hepatocyte secretory factors) for alleviating and/or protecting againstneuronal damage (e.g., resulting from stroke), and methods of usethereof.

BACKGROUND

Cerebral ischemia or ischemic stroke induces neuronal injury and death.As adult neurons possess a limited capacity of protection againstischemic injury, infarcted brain is often replaced with fibrotictissues, resulting in permanent impairment of nervous functions. Onedesired therapeutic approach is to protect the brain from ischemicinjury during the acute phase of stroke, minimizing the loss of neurons.However, few effective therapies have been established for such apurpose in prior research.

SUMMARY OF THE INVENTION

The present invention provides methods, compositions, and systems fortreating a subject experiencing, at risk for, with, suffering from, orsuspected of having cerebral ischemia using hepatocyte secretory factors(e.g., FGF21, RELMγ, TFF3). In some embodiments, the present inventionprovides administering hepatocyte secretory factors to a subject totreat cerebral ischemia. In some embodiments, the present inventionprovides administering hepatocyte secretory factors whose serumconcentration is increased in response to cerebral ischemia. In someembodiments, administration of hepatocyte secretory factors reducesischemic injury in the subject. In some embodiments, administration ofhepatocyte secretory factors exerts a neuroprotective effect. In someembodiments, administration of hepatocyte secretory factors results inreduction in cerebral infarction. In some embodiments, compositions areadministered before, during, and/or after a stroke or other cause ofcerebral infarction or other neuronal injury.

In some embodiments, the present invention provides methods of treatinga subject experiencing cerebral ischemia, the effects of cerebralischemia, or conditions similar to cerebral ischemia comprising:administering a composition to the subject, wherein the compositioncomprises at least one isolated hepatocyte secretory factor. In someembodiments, the serum concentration of the secretory factor isincreased in response to cerebral ischemia (e.g., FGF21, RELMγ, TFF3).In certain embodiments, the administration reduces cerebral injury inthe subject. In some embodiments, administration of secretory factors(e.g., FGF21, RELMγ, TFF3) exerts a neuroprotective effect. In someembodiments, secretory factors are administered in a combination thatexerts a neuroprotective effect, results in reduction in cerebralinfarction, and/or reduces ischemic injury. In some embodiments, the atleast one isolated hepatocyte secretory factor is selected from thegroup consisting of: hepatocyte secreted proteins including fibroblastgrowth factor 21 (FGF21) or a biologically active fragment or variantthereof, resistin like molecule γ (RELMγ) or a biologically activefragment or variant thereof, and trefoil factor 3 (TFF3) or abiologically active fragment or variant thereof.

In certain embodiments, the at least one isolated hepatocyte secretoryfactor is administered at a dosage of 1 μg/kg-500 mg/kg (e.g., 1 μg/kg .. . 5 μg/kg . . . 10 μg/kg . . . 20 μg/kg . . . 50 μg/kg . . . 100 μg/kg. . . 200 μg/kg . . . 500 μg/kg . . . 1 mg/kg . . . 2 mg/kg . . . 5mg/kg . . . 10 mg/kg . . . 20 mg/kg . . . 50 mg/kg . . . 100 mg/kg . . .200 mg/kg . . . 500 mg/kg). In certain embodiments, the at least oneisolated hepatocyte secretory factor is administered at a dosage ofabout 1.0 mg to 1000 mg (e.g., 1.0 mg . . . 2.0 mg . . . 5.0 mg . . . 10mg . . . 20 mg . . . 50 mg . . . 100 mg . . . 200 mg . . . 500 mg . . .1000 mg). In some embodiments, the at least one isolated hepatocytesecretory factor comprises two, three, four, five, or more isolatedhepatocyte secretory factors.

In some embodiments, the subject is a human and the at least oneisolated hepatocyte secretory factor is a human hepatocyte secretoryfactor or active fragment or variant thereof. The at least one isolatedhepatocyte secretory factor can be administered intravenously, orally,transdermally, surgically (e.g., direct administration to the brain), orby any other suitable route of administration.

In certain embodiments, the subject is someone without cerebral ischemictissue injury, but is at risk for such injury (e.g., the composition isadministered prophylacticly to prevent anticipated ischemic injury). Insome embodiments, the subject is experiencing a stroke. In someembodiments, the subject had a stroke in the recent past.

In some embodiments, compositions comprising hepatocyte secretoryfactors (e.g., FGF21, RELMγ, TFF3) are administered with othertherapeutic composition to exert a neuroprotective effect and/or treat,prevent, alleviate cerebral infarction.

In particular embodiments, the subject is a mammal. In otherembodiments, the subject is a human. In some embodiments, the subject isdiagnosed as having cerebral ischemia. In some embodiments, the subjectis suspected of having cerebral ischemia. In some embodiments, thesubject is at risk of having cerebral ischemia.

In some embodiments, the present invention provides a compositioncomprising one or more isolated hepatocyte secretory factors configuredfor pharmaceutical administration to a subject experiencing cerebralischemia. In some embodiments, the one or more isolated hepatocytesecretory factors are selected from the group comprising:

FGF21 or a biologically active fragment or variant thereof, RELMγ or abiologically active fragment or variant thereof, and TFF3 or abiologically active fragment or variant thereof. In some embodiments,pharmaceutical administration is by oral, parenateral, topical,intravenous, transmucosal, surgical, and/or inhalation routes. In someembodiments, a composition further comprises a physiologically tolerablebuffer. In certain embodiments, the present invention provides smallmolecules that cause an increase in one or more of the above isolatedhepatocyte secretory factors.

In some embodiments, the present invention provides methods ofpreventing cerebral ischemia in a subject at risk for experiencingcerebral ischemia comprising: administering a composition to saidsubject, wherein said composition comprises at least one isolatedhepatocyte secretory factor whose serum concentration is increased inresponse to cerebral ischemia. In other embodiments, at least oneisolated hepatocyte secretory factor whose serum concentration isincreased in response to cerebral ischemia is selected from the groupcomprising: FGF21 or a biologically active fragment or variant thereof,RELMγ or a biologically active fragment or variant thereof, and TFF3 ora biologically active fragment or variant thereof. In other embodiments,the administration of said composition exerts a neuroprotective effect.

DESCRIPTION OF THE FIGURES

FIG. 1 shows relative levels of mRNA transcription from genes encodinghepatocyte secreted proteins DEFB1, FGF21, KISS1, RELMγ, SAA1, SAA2, andTFF3 in hepatocytes in cerebral ischemia. For each gene, the time zerorepresents the sham control level. The “relative expression” was definedas the ratio of the mRNA transcription level at a given time to the shamcontrol level. The open squares in each panel represent the relativeexpression of the 13 actin gene.

FIG. 2 shows images of slices of mouse cerebrum for identification ofsecreted factors effective for neuroprotection in cerebral ischemia.Each column represents specimen slices from different locations of themouse cerebrum with administration of albumin or a secreted factor.Specimens were stained with 1% TTC/PBS. Darker regions represents intactbrain, and the white represents brain infarcts. The scale is for allpanels.

FIG. 3 shows a graphic representation of the neuroprotective effect ofsecreted factors FGF21, RELMγ, and TFF3 at 24 hrs of cerebral ischemia.The % represents volume fraction of cerebral infarcts with reference tothe total volume of the cerebrum.

FIG. 4 shows a plot of gripping forces of the left and right forelimbsof mice with right hemispheric cerebral ischemia with administration ofalbumin or a secreted factor.

FIG. 5 shows histograms of measured gripping forces of the left andright forelimbs of mice with right hemispheric cerebral ischemia withadministration of albumin or a secreted factor.

DETAILED DESCRIPTION

Adult neurons possess a limited capacity of protection against ischemicinjury. In the event of cerebral ischemia, non-neuronal cells are ofteninvolved in neuroprotection, contributing to minimization of neuronalinjury and death. Experiments conducted during development ofembodiments of the present invention demonstrated that hepatocytes wereactivated in response to cerebral ischemia to contribute toneuroprotection. These cells upregulate genes encoding secreted proteinsincluding defensin beta 1 (DEFB1), fibroblast growth factor 21 (FGF21),kisspeptin 1 (KISS1), resistin like molecule γ (RELMγ), and trefoilfactor 3 (TFF3) in cerebral ischemia. Functional screening testsidentified three secreted proteins, including FGF21, RELMγ, and TFF3,that exerted a neuroprotective effect. Administration of mouserecombinant FGF21, RELMγ, or TFF3 to mice with cerebral ischemiaresulted in a significant reduction in cerebral infarction andimprovement of the gripping strength of the impaired forelimb. Theseobservations indicate that the secreted factors FGF21, RELMγ, and TFF3upregulated in hepatocytes in response to stroke provide utility inalleviating brain ischemic injury.

Depending on species, FGF21 is generally a 208-210 amino acid proteinbelonging to the FGF family that includes 22 members. This protein isprimarily expressed in the liver and, to a lesser degree, in the thymusand adipose tissue (Nishimura et al., 2000; Ryde'n 2009; hereinincorporated by reference in their entireties). The amino acid sequence(SEQ ID NO:1) and the nucleic acid sequence (SEQ ID NO:2) of human FGF21are shown in the sequence listing. While the majority of FGF membersplay a role in regulating cell proliferation and differentiation, FGF21together with FGF 19 has been reported to regulate glucose and lipidmetabolisms (Itoh and Ornitz, 2004; Dostálová et al., 2009; hereinincorporated by reference in their entireties). FGF21 mediates thesemetabolic activities via interacting with FGF Receptor 2α, resulting inactivation of the ERK1/2 and Akt signaling pathways (Ibrahimi et al.,2004; Kharitonenkov et al., 2005; Mohammadi et al., 2005; Zhang et al.,2006; herein incorporated by reference in their entireties). Thesesignaling pathways may be responsible for FGF21-induced cell activities;although, the present invention is not limited to any particularmechanism of action and an understanding of the mechanism of action isnot necessary to practice the present invention.

RELMγ, also known as resistin like γ (Retnlg), is a secretedcysteine-rich protein (117 amino acids, ˜7 kDa) belonging to the RELMfamily, which is composed of RELMα and RELMβ in addition to RELMγ(Gerstmayer et al., 2003; Schinke et al., 2004; Chumakov et al., 2004;herein incorporated by reference in their entireties). These moleculesdisplay considerable sequence homology to resistin, a secreted proteinthat negatively regulates the sensitivity of cells to insulin (Steppanet al., 2001; Nair et al., 2006; herein incorporated by reference intheir entireties). RELMγ is primarily expressed in the lung, spleen,pancreas, small and large intestines, and bone marrow (Shojima et al.,2005; Gerstmayer et al., 2003; Schinke et al., 2004; Chumakov et al.,2004; herein incorporated by reference in their entireties). Thismolecule may form homodimers and heterodimers with RELMβ, and has beenreported to stimulate the proliferation and differentiation ofpromyelocytic cells in the rat (Schinke et al., 2004; hereinincorporated by reference in its entirety), and suspected to mediateinflammatory responses and cause insulin resistance (Nair et al., 2006;Shojima et al., 2005; herein incorporated by reference in theirentireties). RELMγ receptors and associated signaling pathways have notbeen identified. It is important to note that, under physiologicalconditions, RELMγ is not expressed in the liver. Experiments conductedduring development of embodiments of the present invention demonstratedthat hepatocyte mRNA transcription for the RELMγ gene was upregulatedfor 4.4 times in cerebral ischemia compared to the sham control. Theseobservations indicate that RELMγ is upregulated in response to cerebralischemia, although the signaling mechanisms remain to be addressed. Insome embodiments of the present invention, RELMβ is administered to asubject in place or, or in conjunction with RELMγ.

TFF3 is a protein expressed primarily in mucus-secreting goblet cells ofthe gastrointestinal tract (Sands and Podolsky 1996; herein incorporatedby reference in its entirety). The amino acid sequence (SEQ ID NO:5) andthe nucleic acid sequence (SEQ ID NO:6) of human TFF3 are shown in thesequence listing. TFF 3 has been shown to contribute to the maintenanceof the mucosal integrity and facilitate mucosal healing and repair aftermechanical and chemical injury (Sands and Podolsky 1996; Wong et al.,1999; herein incorporated by reference in their entireties).

Experiments conducted during development of embodiments of the presentinvention demonstrated the contribution of these factors toneuroprotection against ischemic injury.

The hepatocyte secretory factors may be administered in combinations oftwo of more factors (e.g., as a single combination or sequentiallyadministered). Exemplary combinations of factors, which can consist ofor comprise these combinations, are shown in Table 1 below:

TABLE 1 Combination 1 FGF21 Combination 2 FGF21 RELMγ Combination 3FGF21 TFF3 Combination 4 TFF3 RELMγ Combination 5 FGF21 RELMγ TFF3Combination 5 RELMγ Combination 5 TFF3In some embodiments, the combinations listed in Table 1 are combinedwith one or more additional hepatocyte secretory factors foradministration to a subject. In some embodiments, the combinationslisted in Table 1 are combined with one or more additional therapeuticagents for administration to a subject.

In certain embodiments, the present invention provides a sequencevariant of a hepatocyte secretory factor, such as FGF21, RELMγ, and/orTFF3, including, for example, a deletion, insertion, mutation, or thelike, that is useful in treating, preventing, or alleviating cerebralischemic injury. Such a sequence variant or fragment is termed a“biologically active fragment or variant” of a hepatocyte secretoryfactor. In some embodiments, a biologically active fragment or variantprovides one or more portions of a hepatocyte secretory factor, such asFGF21, RELMγ, and/or TFF3. In some embodiments, a biologically activeportions or fragments of a hepatocyte secretory factor, such as FGF21,RELMγ, and/or TFF3 is useful in treating, preventing, or alleviatingcerebral ischemic injury. In some embodiments, biologically activefragment or variant of a hepatocyte secretory factor includes,polypeptides having at least 70% identity to FGF21, RELMγ, and/or TFF3(e.g., have a sequence identity to FGF21, RELMγ, and/or TFF3 thatis >99% . . . >98% . . . >95% . . . >90% . . . >85% . . . >80% . .. >75% . . . >70%). A biologically active fragment or variant of ahepatocyte secretory factor can include a polypeptide that has at least20 amino acids having at least 70% identity to a contiguous portion ofFGF21, RELMγ, or TFF3. For example, a biologically active fragment orvariant of a hepatocyte secretory factor includes a polypeptidecomprising a hepatocyte secretory factor, such as FGF21, RELMγ, and/orTFF3 with an N-terminal deletion of up to 100 amino acids. In anotherexample, a biologically active fragment or variant of a hepatocytesecretory factor includes a polypeptide comprising a hepatocytesecretory factor, such as FGF21, RELMγ, and/or TFF3 with a C-terminaldeletion of up to 100 amino acids. One can determine if varioustruncations or variants of the secretory factors function in aneuroprotective capacity by substituting in these truncations orvariants in the Examples below.

In some embodiments, the present invention provides one or moreconserved portions of a hepatocyte secretory factor, such as FGF21,RELMγ, and/or TFF3. The present invention also contemplates sequencesthat have at least 70% (e.g., 70% . . . 75% . . . 80% . . . 85% . . .90% . . . 95%, 96%, 97%, 98%, 99%, or 99.5%) sequence identity to ahepatocyte secretory factor, such as FGF21, RELMγ, and/or TFF3 and/orexhibits substantially the same, or similar, activity (e.g., treating,preventing, or alleviating cerebral ischemic injury). For example, oneor two amino acids may be changed (or one or two codons changed in thenucleic acid) such that a sequence differing by one or two amino acidresidues is generated. In some embodiments, multiple amino acids inthese sequences are changed (or codons in nucleic acids coding thesesequences) while maintaining similar secondary structure and/or tertiarystructure, and maintaining activity. Changes to the amino acid sequencemay be generated by changing the nucleic acid sequence encoding theamino acid sequence.

Nucleic acid encoding a variant of a given portion of these sequencesmay be prepared by methods known in the art. These methods include, butare not limited to, preparation by site-directed (oroligonucleotide-mediated) mutagenesis, PCR mutagenesis, and cassettemutagenesis of an earlier prepared nucleic acid encoding a hepatocytesecretory factor, such as FGF21, RELMγ, and/or TFF3. Site-directedmutagenesis is a preferred method for preparing substitution variants.This technique is well known in the art (see, e.g., Carter et al.Nucleic Acids Res. 13: 4431-4443 (1985) and Kunkel et. al., Proc. Natl.Acad. Sci. USA 82: 488 (1987), both of which are hereby incorporated byreference).

Briefly, in carrying out site directed mutagenesis of DNA, the startingDNA is, for example, altered by first hybridizing an oligonucleotideencoding the desired mutation to a single strand of such starting DNA.After hybridization, a DNA polymerase is used to synthesize an entiresecond strand, using the hybridized oligonucleotide as a primer, andusing the single strand of the starting DNA as a template. Thus, theoligonucleotide encoding the desired mutation is incorporated in theresulting double-stranded DNA.

PCR mutagenesis is also suitable for making amino acid sequence variantsof a hepatocyte secretory factor, such as FGF21, RELMγ, and/or TFF3(see, e.g., Vallette et. al., Nuc. Acids Res. 17: 723-733 (1989), herebyincorporated by reference). Briefly, when small amounts of template DNAare used as starting material in a PCR, primers that differ slightly insequence from the corresponding region in a template DNA can be used togenerate relatively large quantities of a specific DNA fragment thatdiffers from the template sequence only at the positions where theprimers differ from the template.

Another method for preparing variants, cassette mutagenesis, is based onthe technique described by Wells et al., Gene 34: 315-323 (1985), herebyincorporated by reference. The starting material is the plasmid (orother vector) comprising the nucleic acid encoding the hepatocytesecretory factor to be mutated. The codon(s) in the starting DNA to bemutated are identified. There should be a unique restrictionendonuclease site on each side of the identified mutation site(s). If nosuch restriction sites exist, they may be generated using theabove-described oligonucleotide-mediated mutagenesis method to introducethem at appropriate locations in the starting polypeptide DNA. Theplasmid DNA is cut at these sites to linearize it. A double-strandedoligonucleotide encoding the sequence of the DNA between the restrictionsites but containing the desired mutation(s) is synthesized usingstandard procedures, wherein the two strands of the oligonucleotide aresynthesized separately and then hybridized together using standardtechniques. This double-stranded oligonucleotide is referred to as thecassette. This cassette is designed to have 5′ and 3′ ends that arecompatible with the ends of the linearized plasmid, such that it can bedirectly ligated to the plasmid. This plasmid now contains the mutatedDNA sequence.

Biologically active fragment or variant of a hepatocyte secretory factorcan be screened in assays known in the art to determine if suchpolypeptides are suitable for use in embodiments of the presentinvention. For example, one can screen such mutants and variants usingthe methods described in Examples (below), by substituting the candidatevariants hepatocyte secretory factor for those described in Example 1and determining if ischemic injury is reduced or prevented.

Alternatively, or additionally, the desired amino acid sequence encodinga polypeptide variant can be determined, and a nucleic acid sequenceencoding such amino acid sequence variant can be generatedsynthetically. Conservative modifications in the amino acid sequences ofhepatocyte secretory factors, or in the nucleic acids encoding suchfactors, may also be made by substituting an amino acid with an aminoacid of the same class. Amino acid residues (e.g., natural and/ornon-natural) are divided into classes based on common side-chainproperties, for example:

(1) hydrophobic: norleucine, met, ala, val, leu, ile;

(2) neutral hydrophilic: cys, ser, thr;

(3) acidic: asp, glu;

(4) basic: asn, gln, his, lys, arg;

(5) residues that influence chain orientation: gly, pro; and

(6) aromatic: trp, tyr, phe.

In some embodiments, the present invention employs variants of FGF21,such as those described in Pat. Pub. 20090305986 and U.S. Pat. No.7,655,627 (both of which are herein incorporated by reference). In someembodiments, the present invention employs the proteins and peptidesdescribed in Pat. Pub. 20110008281-A1, or nucleic acids encoding suchproteins and peptides. Examples of FGF21, RELMγ, and TFF3 polypeptidesequences, and nucleotide sequences encoding them, can be found in thesequence listing as SEQ ID NOs:1-6. Polypeptide sequences comprising,consisting of, and/or consisting essentially of SEQ ID NOs:1, 3, and/or5, biologically active variants (e.g. >70% identity) and/or fragmentsthereof find use in embodiments described herein. Nucleotide sequencescoding for polypeptides comprising, consisting of, and/or consistingessentially of SEQ ID NOs:1, 3, and/or 5, biologically active variants(e.g. >70% identity) and/or fragments thereof find use in embodimentsdescribed herein. Nucleotide sequences comprising, consisting of, and/orconsisting essentially of SEQ ID NOs:2, 4, and/or 6, fragments thereof,and/or variants thereof find use in embodiments described herein.

In some embodiments, the present invention provides compositions andmethods for prevention, treatment and/or alleviation of cerebralischemic injury (e.g., cerebral infarction) resulting from stroke. Insome embodiments, compositions of the present invention are provided asa pharmaceutical agent. When used for the purposes described herein,said pharmaceutical agent may be administered via any desired oral,parenateral, topical, intravenous, transmucosal, surgical, and/orinhalation routes. The pharmaceutical agent may be administered in theform of a composition which is formulated with a pharmaceuticallyacceptable carrier and optional excipients, flavors, adjuvants, etc. inaccordance with good pharmaceutical practice.

In some embodiments of the present invention, compositions areadministered to a patient alone or in combination with other therapies,pharmaceuticals, supplements, and/or a specified diet, or inpharmaceutical compositions where it is mixed with excipient(s) or otherpharmaceutically acceptable carriers. In some embodiments of the presentinvention, the pharmaceutically acceptable carrier is pharmaceuticallyinert. In other embodiments of the present invention, compositions maybe administered alone.

Depending on the goal of administration (e.g. type and severity ofcondition, duration of treatment, etc.), compositions may be formulatedand administered systemically or locally (e.g., in the brain).Techniques for formulation and administration may be found in the latestedition of “Remington's Pharmaceutical Sciences” (Mack Publishing Co,Easton Pa.). Suitable routes may, for example, include oral ortransmucosal administration; as well as parenteral delivery, includingintramuscular, subcutaneous, intramedullary, intrathecal,intraventricular, intravenous, intraperitoneal, or intranasaladministration.

In some embodiments, compositions may be in the form of a solid,semi-solid or liquid dosage form: such as tablet, capsule, pill, powder,suppository, solution, elixir, syrup, suspension, cream, lozenge, pasteand spray containing the first and second agents formulatedappropriately to provide the desired time-release profile. As thoseskilled in the art would recognize, depending on the chosen route ofadministration, the composition form is selected.

In some embodiments, the pharmaceutical agent may be administered insingle or multiple doses. The particular route of administration and thedosage regimen will be determined by one of skill, in keeping with thecondition of the individual to be treated and said individual's responseto the treatment. The present invention also provides pharmaceuticalagents in a unit dosage form for administration to a subject, comprisingpharmaceutical agents and one or more nontoxic pharmaceuticallyacceptable carriers, adjuvants or vehicles. The amount of the activeagents (e.g. FGF21, RELMγ, TFF3, etc.) that may be combined with suchmaterials to produce a single dosage form will vary depending uponvarious factors, as indicated above. A variety of materials can be usedas carriers, adjuvants, and vehicles in the composition of theinvention, as available in the pharmaceutical art.

In some embodiments, compositions of the present invention areco-administered with other therapeutics for treatment, prevention, oralleviation of cerebral ischemia or other neuronal damage or disorders.In some embodiments, active agents (e.g. FGF21, RELMγ, TFF3, etc.) areco-administered with any suitable agents (e.g., therapeutics,nutriceutical, pharmaceuticals, etc.), for treatment, prevention,symptom reduction, easing side effects of treatments, deducing druginteractions, etc.

EXAMPLES

The following Examples are presented in order to provide certainexemplary embodiments of the present invention and are not intended tolimit the scope thereof.

Example 1 Neuroprotective Effect of Stroke-Induced Secretory Proteins

Cerebral Ischemia.

Cerebral ischemia was induced in the mouse by permanently ligating theright middle cerebral artery with 2-hr ligation of both common carotidarteries (Buchan A M, et al., 1992; herein incorporated by reference inits entirety). C57BL/6J mice were anesthetized by intraperitonealinjection of ketamine (100 mg/kg) and xylazine (10 mg/kg). A skinincision was made between the right eye and the external auditory canal.A 2 mm hole was drilled on the temporal bone near the zygomatic arch.The middle cerebral artery was permanently ligated with a silk suturethread at the first bifurcation above the zygomatic arch. Immediatelyfollowing the ligation of the middle cerebral artery, both commoncarotid arteries were ligated for 2 hrs. Age- and gender-matched micewere used as controls with sham operation with identical proceduresexcept that the middle cerebral and both carotid arteries were notligated. The Institutional Animal Care and Use Committee approved allexperimental procedures used in this investigation.

cDNA Microarray.

cDNA microarray analyses were conducted to test the gene expressionprofile in hepatocytes by using the Illumina whole-genome Mouse WG-6 v2Expression Bead Chip with 45,200 transcripts. Hepatocytes were isolatedfrom the liver of mice with cerebral ischemia and sham operation at day1, 3, 5, and 10 by Percoll-mediated centrifugation. Total RNA wasprepared from the isolated hepatocytes by using the TRIzol reagent kitof Invitrogen. cDNA and cRNA syntheses, cRNA chip hybridization, anddata scanning were carried out according to the manufacturer'sinstruction. An average number of 41+/−8 spots were used for each gene.The target signal intensity values of all spots for each gene wereaveraged and used for data analyses. The acquisition and initialquantification of array images were achieved using the Illumina GenomeStudio Gene Expression Module v1.0 software. The target signal intensitywas normalized with reference to the median intensity value. Criteriafor selecting differentially expressed genes in the hepatocytes of micewith cerebral ischemia were set at a 3-fold change compared to the shamcontrol at a given time and a detection p-value at p<0.05 based on thecomparison of the target signal with the negative control. Upregulatedgenes encoding secreted proteins were further tested and analyzed forselecting neuroprotective factors.

Identification of Secreted Factors Effective for Neuroprotection.

To identify factors effective for neuroprotection from the population ofupregulated genes encoding secreted proteins, the recombinant form ofeach upregulated secreted factor was administered to mice with cerebralischemia and tested changes in the volume fraction of cerebral infarcts.A selected recombinant protein was injected IV at a dose of 50 ng/gmtwice per day starting immediately following the induction of cerebralischemia. Mouse serum albumin at the same dose was used as a control.The volume fraction of cerebral infarcts was tested by the TTC assay at24 hrs post cerebral ischemia. A protein was considered effective forneuroprotection when its administration resulted in a significantreduction in the volume fraction of cerebral infarcts compared to thealbumin control.

Measurement and Analysis of Cerebral Infarcts.

The degree of cerebral injury was evaluated based on the volume fractionof cerebral infarcts with reference to the total volume of the cerebrum.The 2,3,5-triphenyltetrazolium chloride (TTC) assay was used forstaining and measuring the intact and infarcted brain at 24 hrs ofcerebral ischemia as described (Liu and Wu, 2010a). A fresh mouse brainwas collected, frozen rapidly at −66° C. for 5 min on a pre-cooledrubber pad, and cut into 1-mm thick slices using a blade with a 1-mmspacer. The brain slices were incubated in 1% TTC/PBS at 37° C. for 30min, fixed in 4% formaldehyde/PBS for 15 min, and photographed with theaid of a surgical microscope.

TTC stains intact brain red and infarcts white. As there exists a widetransition zone with a gradient of red intensity from the red to thewhite region, it is often difficult to accurately and reproduciblyidentify the boundary of the infarct, resulting in intra- andinter-observer measurement errors. To resolve this problem, astatistical model was established to define the cerebral infarctboundary in TTC-stained specimens based on RGB-formatted images. In thismodel, the red, green, and blue color intensities were measured fromimage regions randomly selected from the intact brain of each testedspecimen. A TTC index was calculated for each image region to representthe red intensity by using the following equation:

TTC index=Red intensity/(Red intensity+Green intensity+Blue intensity).

An equal intensity for all three colors, except for zero, gives a TTCindex 1/3, representing the white color. A definite red intensity with azero green and blue intensity gives a TTC index 1, representing a purered color. The TTC index for all measured image regions from the intactbrain of each specimen was analyzed to test the statisticaldistribution. The TTC index follows a normal distribution pattern inTTC-stained brain specimens. The mean and standard deviation of the TTCindex were calculated for each specimen. A TTC boundary index foridentifying the red-white or intact-infarcted brain boundary was definedbased on the statistical principle of population identification:

Boundary index=Mean of TTC index−1.96×standard deviation.

This index represents the boundary of a cerebral infarct. An imageregion with a TTC index>the boundary index was considered a portion ofthe intact brain, whereas an image region with a TTC index<the boundaryindex was considered a portion of the infarct. The total area of the redor white regions represents the area of the intact or infarcted brain,respectively. The volume fraction of the intact and infarcted brain wascalculated with reference to the total volume of the cerebrum.

Measurement and Analysis of Mouse Forelimb Gripping Strength.

Cerebral ischemia induces impairment of the motor function of skeletalmuscles. In this investigation, the forelimb gripping force was measuredand analyzed to assess the degree and progression of cerebral injury inresponse to ischemic insults with and without a treatment with asecreted factor. A torque device was developed for testing the mouseforelimb gripping force (SEE FIG. 1). During the test, a mouse wastail-lifted, one of the mouse forelimbs was wrapped with a piece of aScotch tape to prevent the limb from gripping, and the other forelimbwas allowed to grip a small ring (1 cm) attached to the arm of a torquesensor (arm length 5 cm). The mouse was tail-pulled slowly in thedirection perpendicular to the arm of the torque sensor until the mousegave up gripping the arm ring. The mouse forelimb gripping force wasrecorded continuously during the test via a data acquisition system. Therecorded data were converted to gripping forces based on calibrationsderived by using 10, 20, and 30 gm weights.

Upregulation of Hepatocyte Genes Encoding Secreted Factors in CerebralIschemia.

Cerebral ischemia induced upregulation of hepatocyte genes encodingsecreted proteins, including defensin beta 1 (DEFB1), fibroblast growthfactor 21 (FGF21), kisspeptin (KISS1), resistin-like molecule γ (RELMγ),serum amyloid A (SAA) 1, SAA2, trefoil factor 3 (TFF3). The maximallevel of mRNA transcription was found at day 1 for the DEFB1, FGF21,KISS1, and RELMγ genes, and at day 5 for the SAM and SAA2 genes postcerebral ischemia (SEE FIG. 2). The TFF3 gene exhibited two episodes oftranscription upregulation at day 1 and 5. The maximal relative level ofmRNA transcription was 3.2, 3.3, 4.4, and 3.1 times of the sham controllevel for the DEFB1, KISS1, RELMγ, and TFF3 genes, respectively, 9.6times for the FGF21 gene, and 25.4 and 15.4 times for the SAA1 and SAA2genes, respectively. In this investigation, we tested theneuroprotective effect of DEFB1, FGF21, KISS1, RELMγ, and TFF3. SAM andSAA2 were not tested, because these factors are known to induceamyloidosis, resulting in impairment of cell and tissue functions(Westermark and Westermark, 2009; herein incorporated by reference inits entirety).

Effect of Secreted Factor Administration on Cerebral Infarction.

To test the neuroprotective effect of DEFB1, FGF21, KISS1, RELMγ, andTFF3, the mouse recombinant form of each protein was administered tomice with cerebral ischemia at the dose of 50 ng/gm twice per daystarting immediately post cerebral ischemia, and tested the effect ofthe administration on the volume fraction of cerebral infarcts at 24 hrsby the TTC assay, while albumin was used as a control agent.Administration of FGF21, RELMγ, or TFF3 resulted in a significantreduction in the volume fraction of cerebral infarcts at 24 hrs, whereasadministration of DEFB1 or KISS1 did not induce a significant change(SEE FIGS. 3 and 4). Thus, FGF21, RELMγ, and TFF3 were identified aspotential neuroprotective factors in this investigation.

Effect of Secreted Factor Administration on the Gripping Strength of theImpaired Forelimb.

Cerebral ischemia induced a significant reduction in the grippingstrength of the opposite forelimb. Experiments were conducted duringdevelopment of embodiments of the present invention to determine whetheradministration of DEFB1, FGF21, KISS1, RELMγ, or TFF3 improved thegripping strength of the impaired forelimb in mice with cerebralischemia. While administration of DEFB1 or KISS1 at the dose of 50 ng/gmtwice per day did not influence the forelimb gripping strength at 24 hrsof cerebral ischemia, administration of FGF21, RELMγ, or TFF3significantly improved the gripping strength of the impaired forelimb(SEE FIGS. 5 and 6). These observations were consistent with the resultspresented in the section above, suggesting that FGF21, RELMγ, and TFF3exerted a protective effect on the motor control function of the centralnerve system in cerebral ischemia.

The liver is a unique organ that possesses a high capacity of protectionand regeneration in mechanical and chemical injuries. Live cells,including hepatocytes, biliary epithelial cells, endothelial cells,Kupffer cells, an Ito cells, are capable of initiating rapidproliferation during liver regeneration (Alison et al., 1998; Fausto andCampbell, 2003; Forbes et al., 2002; Michalopoulos and DeFrances, 1997;Taub, 2004; herein incorporated by reference in their entireties).Observations from this investigation suggest that the liver cells arenot only responsible for liver protection and regeneration, but alsocontribute to protection of ischemic brain tissues. These observationsprovide a basis for investigations of liver responses to brain injuries.

All publications and patents mentioned in the present application and/orlisted below are herein incorporated by reference. Various modificationand variation of the described methods and compositions of the inventionwill be apparent to those skilled in the art without departing from thescope and spirit of the invention. Although the invention has beendescribed in connection with specific preferred embodiments, it shouldbe understood that the invention as claimed should not be unduly limitedto such specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention that are obvious to thoseskilled in the relevant fields are intended to be within the scope ofthe following claims.

REFERENCES

-   Alison M, Golding M, Lalani El-N & Sarraf C. Philos Trans R Soc Lond    B Biol Sci 353, 877-894, 1998.-   Buchan A M, et al., Stroke 23:273-279, 1992-   Chumakov A M, Kubota T, Walter S, Koeffler H P (2004). Oncogene 23:    3414-3425-   Dostálová I, Haluziková D, and Haluzik M. Physiol Res 58, 1-7, 2009.-   Fausto N, and Campbell J S (2003). Mech Dev 120, 117-130.-   Forbes S, Vig P, Poulsom R, Thomas H, and Alison M. J. Pathol. 197,    510-518, 2002.-   Gerstmayer B, Kusters D, Gebel S et at (2003). Genomics 81:588-595-   Ibrahimi O A, Zhang F, Hrstka S C, Mohammadi M, Linhardt R T.    Biochemistry 43: 4724-4730, 2004.-   Itoh N, Ornitz D M. Trends Genet 20: 563-569, 2004.-   Kharitonenkov A, Shiyanova T L, Koester A, Ford A M, Micanovic R,    Galbreath E J, Sandusky G E, Hammond L J, Moyers J S, Owens R A,    Gromada J, Brozinick J T, Hawkins E D, Wroblewski V J, Li D S,    Mehrbod F, Jaskunas S R & Shanafelt A B (2005). J Clin Invest 115,    1627-1635.-   Liu S Q, and Wu Y H. Frontiers in Bioscience (Elite Ed) 2:711-24,    2010a.-   Liu S Q. and Wu Y H. Current Topics in Biochemical Research, 2010b,    in press.-   Shu Q. Liu, Yupeng Ren, Brandon Tefft, Yi-Ning Wu, Charley Liu, Yan    Chun Li, Li-Qun Zhang, Barry Walker, Harry R. Phillips, Yu H. Wu*.    2010, submitted.-   Michalopoulos G K & DeFrances M C (1997). Science 276, 60-66.-   Mohammadi M, Olsen S K, Goetz R. Curr Opin Struct Biol 15: 506-516,    2005.-   Nair M G, Guild K J, and Ards D. J. Immunol. 2006; 177; 1393-1399-   Nishimura T, Nakatake Y, Konishi M, and Itoh N. Biochim Biophys Acta    1492: 203-206, 2000.-   Ryde'n M. Cell. Mol. Life Sci. 66:2067-2073, 2009.-   Sands B E, and Podolsky D K. Annu Rev. Physiol. 58:253-273, 1996.-   Schinke T, Haberland M, Jamshidi A et at (2004). Biochem Biophys Res    Commun 314:356-362-   Shojima N., Ogihara T. Inukai K., Fujishiro M., Sakoda H., Kushiyama    A., Katagiri H., M. Anai M., Ono H., Fukushima Y., Horike N.,    Viana A. Y. I., Uchijima Y., Kurihara H., Asano T. Diabetologia 48:    984-992, 2005.-   Steppan C M, Bailey S T, Bhat S, Brown E J, Banerjee R R, Wright C    M, Patel H R, Ahima R S, Lazar M A. Nature. 2001 Jan. 18;    409(6818):307-12.-   Taub R (2004). Nat Rev Mol Cell Biol 5, 836-847.-   Westermark G T, Per Westermark. FEBS Letters 583: 2685-2690, 2009.-   Wong W M, Poulsom R, Wright N A. Gut 44:890-895, 1999.-   Zhang X, Ibrahimi O A, Olsen S K, Umemori H, Mohammadi M, Ornitz    D M. J Biol Chem 281: 15694-15700, 2006.

We claim:
 1. A method of treating a subject experiencing cerebralischemia comprising: administering a composition to said subject,wherein said composition comprises at least one isolated hepatocytesecretory factor whose serum concentration is increased in response tocerebral ischemia.
 2. The method of claim 1, wherein said at least oneisolated hepatocyte secretory factor whose serum concentration isincreased in response to cerebral ischemia is selected from the groupcomprising: FGF21 or a biologically active fragment or variant thereof,RELMγ or a biologically active fragment or variant thereof, and TFF3 ora biologically active fragment or variant thereof.
 3. The method ofclaim 1, wherein administration of said composition reduces ischemicinjury in said subject.
 4. The method of claim 1, wherein administrationof said composition exerts a neuroprotective effect.
 5. The method ofclaim 1, wherein administration of said composition results in reductionin cerebral infarction.
 6. The method of claim 1, wherein said subjectis a mammal.
 7. The method of claim 6, wherein said subject is a human.8. The method of claim 7, wherein said at least one isolated hepatocytesecretory factor is a human hepatocyte secretory factor or fragment orvariant thereof.
 9. The method of claim 8, wherein said human hepatocytesecretory factor is selected from the group comprising: human FGF21 or abiologically active fragment or variant thereof, human RELMγ or abiologically active fragment or variant thereof, and human TFF3 or abiologically active fragment or variant thereof.
 10. A compositioncomprising one or more isolated hepatocyte secretory factors configuredfor pharmaceutical administration to a subject experiencing cerebralischemia.
 11. The composition of claim 10, wherein said one or moreisolated hepatocyte secretory factors are selected from the groupcomprising: FGF21 or a biologically active fragment or variant thereof,RELMγ or a biologically active fragment or variant thereof, and TFF3 ora biologically active fragment or variant thereof.
 12. The compositionof claim 11, wherein said one or more isolated hepatocyte secretoryfactors are selected from the group consists essentially of: FGF21 or abiologically active fragment or variant thereof, RELMγ or a biologicallyactive fragment or variant thereof, and TFF3 or a biologically activefragment or variant thereof.
 14. The composition of claim 12, whereinsaid one or more isolated hepatocyte secretory factors are selected fromthe group consists of: FGF21 or a biologically active fragment orvariant thereof, RELMγ or a biologically active fragment or variantthereof, and TFF3 or a biologically active fragment or variant thereof.15. The composition of claim 10, wherein pharmaceutical administrationis by oral, parenateral, topical, intravenous, transmucosal, surgical,and/or inhalation routes.
 16. The composition of claim 10, furthercomprising a physiologically tolerable buffer.
 17. A method ofpreventing cerebral ischemia in a subject at risk for experiencingcerebral ischemia comprising: administering a composition to saidsubject, wherein said composition comprises at least one isolatedhepatocyte secretory factor whose serum concentration is increased inresponse to cerebral ischemia.
 18. The method of claim 17, wherein saidat least one isolated hepatocyte secretory factor whose serumconcentration is increased in response to cerebral ischemia is selectedfrom the group comprising: FGF21 or a biologically active fragment orvariant thereof, RELMγ or a biologically active fragment or variantthereof, and TFF3 or a biologically active fragment or variant thereof.19. The method of claim 17, wherein administration of said compositionexerts a neuroprotective effect.